Lewis L. Coriell

Some comments on herpetic infection in children with special emphasis on unusual clinical manifestations

Summary A review of the epidemiology ofherpetic infection has illustrated areas in which further information is still needed. The clinical presentations were made with the objective of drawing the attention of clinicians to some of the more unusual manifestations of this infection so that it may be considered more regularly in mucous membrane and skin lesions and encephalitis in children. The relation of maternal immunity to fatal herpetic infection of the newborn infant is a further area for investigation.

Differential susceptibility of epithelial cells and fibroblasts of human skin to freeze injury: Report on an improved method for storage of human skin at −196°C*

Summary Fresh human skin placed in plasma clot culture shows outgrowth of epithelial cells within 3 days followed 3 to 6 days later by fibroblasts. Plasma clot culture of human skin pretreated in medium containing 10% glycerol at 4°C, frozen, and thawed showed outgrowth of fibroblasts only. Using this increased susceptibility of epithelial cells to freeze injury as a criterion, we developed a freezing method in which skin is pretreated with 20 to 30% glycerol at 4°C for 1 to 2 hrs before freezing. Human skin preserved in liquid nitrogen by this method grew out in plasma clot culture very much like fresh skin.

PRESERVATION OF CELL CULTURES BY FREEZING IN LIQUID NITROGEN VAPOR.

Summary Nine continuous, 2 diploid and one primary cell line were successfully preserved by freezing in liquid nitrogen vapor at cooling rates of 1.4°C/min to 43°C/min, but poor survival was observed when cells were frozen by immersion directly in liquid nitrogen. The cells were frozen directly in a liquid nitrogen refrigerator by placing the ampuls at different distances below the lip, demonstrating that elaborate and expensive cell preserving equipment is not required. The plating efficiency technique was shown to be the most sensitive indicator of cell damage.

Viability of Cell Cultures Following Extended Preservation in Liquid Nitrogen

Summary Twenty-two frozen batches of 16 different cell lines certified by the Cell Culture Collection Committee were successfully stored in liquid nitrogen over an interval of 2.5 to 4.5 years. Confluent cell growth obtained in milk dilution bottles and roller .tubes prepared from thawed aimpules of frozen cells clearly demonstrated the high viability of these cell lines. The studies indicated that cells harvested and frozen under optimal conditions retained high viability during prolonged storage in liquid nitrogen, but that damaged cells as shown by initial low viability may be expected to show more variable results.

ETIOLOGICAL STUDIES ON BOVINE LYMPHOSARCOMA

European workers believe that the incidence of lymphosarcoma, or “leukosis” t of cattle, has increased since World War I1 in the cattle populations of Denmark, Sweden, and Germany. They also believe that “leukosis” is a slowly advancing infectious disease with a long incubation period in which the initial sign is an increase in the absolute lymphocyte An infectious etiology for lymphosarcoma has also been suggested by Goetze et al. 1956’; by the reported isolation of a virus from “leukosis” cattle by Thorell, 1957,’O Montemagno et ul., 1957,11 and Papparella, 195912; by the cattle transmission experiments of Rosenberger, 196lS; and by the recent reports of McKercher, 1962,13-14 Jarrett, 1962,16 and Sorenson, 1962.I6-l7 However, proof that an infectious agent is involved in the etiology of bovine lymphosarcoma is still lacking. Our group has recently reported on attempts to characterize lymphosarcoma with regard to clinical manifestations, pathologic alterations and familial distribution of cases in high incidence herds.18 Analysis of pedigree data from cattle with lymphosarcoma in multiple-case herds indicates that the disease occurs in related animals. Such a familial aggregation of cases is suggestive of genetic susceptibility to lymphosarcoma and/or vertical transmission of an infectious agent. We have been attempting to exploit the clinical, hematological, and epidemiological data in developing criteria for the selection of cattle to be used in etiological studies of lymphosarcoma. Cattle are considered to be “normal” or “noninfected” 1 if they: 1. Show no clinical signs of lymphosarcoma. 2. Are derived from herds with no past history of lymphosarcoma. 3. Are derived from herds not showing generalized lymphocytosis on re-

Human skin grafted upon the chorioallantois of the chick embryo for virus cultivation.

Summary and Conclusions (1) Human skin may readily be grafted onto the chorioallantoic membrane of embryonated eggs. (2) The prepuce removed by usual surgical circumcision is a satisfactory source of adequate amounts of skin. (3) Penicillin and streptomycin obviate most bacterial contamination. (4) Grafted human skin may be passed serially from egg to egg at weekly intervals and remain viable. (5) Herpes simplex and vaccinia grow readily on grafted human skin, with the formation of characteristic inclusion bodies. (6) A successful inoculation with herpes zoster confirms the previous single successful cultivation of this virus outside of the human body.

The effect of prolonged storage of cell cultures in dimethyl sulfoxide and glycerol prior to freezing

Summary Two tissue culture cell lines, HeLa and WI-38, were stored in the presence of 5% DMSO or 5% glycerol for intervals of 24 to 168 hrs at 4 and 37°C to test the toxic effects of these additives on the cells. The studies demonstrated the superiority of storage at 4°C. Viability was reduced after 72 hrs of storage of HeLa cells at 4°C and 24 hrs at 4°C was found to be the maximal storage time without toxic effects. During this time interval no demonstrable loss of viability was observed as monitored by the cell permeability and cell adhesion tests in either the frozen or unfrozen cells. Cell damage of the more delicate WI-38 cells was observed after 24 hrs at 4°C in both the prefreeze and post-thaw cultures; 6 hrs was the maximal storage period at 4°C without loss of viability. We recommend from the results of these studies that the cell lines be stored at 4°C when they cannot be immediately frozen after ampulizing. It is suggested that the more delicate diploid and primary cell cultures must be frozen within 3 to 6 hrs of ampulizing. Although the more hardy established cell lines may be left overnight at 4°C and successfully frozen on the next day with little loss in viability, it is advisable to freeze them within several hours after ampulizing to minimize cell damage.

In vitro preservation of functioning rabbit hearts in a depolarized state at 4°C*†

Summary Tissue culture media of various types have been devised in the past few years to support the growth of living cells for many generations vitro . We have made modifications in one tissue culture medium (Eagles minimum essential medium, MEM) so that it will support the functional viability of rabbit hearts stored for 48 hrs at 4°C. Rabbit hearts perfused with Eagles MEM with added KCl to give a final concentration of 13 to 15 mEq per liter of K + stop contracting within 1 min. Perfusion with the high K + medium is then continued at 4°C. After a series of 40 experiments it has been established that rabbit hearts perfused with Eagles MEM containing 143 mEq of Na + per liter, 13 to 15 mEq of K + per liter, 9.8 mEq of Ca ++ per liter, and 0.2% glucose equilibrated with 95% O 2 -5% CO 2 can be preserved at 4°C for 24 to 48 hrs with 100% recovery of function as measured by the force of contraction.

Occurrence of penicillin-resistant staphylococci in patients receiving penicillin orally for prophylaxis of recurrences of rheumatic fever

Abstract The occurrence of penicillin-resistant staphylococci has been studied in throat cultures of children attending rheumatic fever clinics who were maintained on a prophylactic regimen with orally administered penicillin, and in some control groups. Among children maintained on this regimen 48 per cent were found to harbor penicillin-resistant staphylococci, and 37 per cent of the strains were of bacteriophage group III. In the two non-rheumatic control groups 8 per cent were positive for penicillin-resistant staphylococci and 5 to 6 per cent were in phage group III. Among children attending the same clinics but not given penicillin prophylaxis the percentages were approximately the same as in these control groups. There was a high degree of association between penicillin resistance and phage group III in the staphylococci isolated from the group receiving penicillin. In the group receiving penicillin there were substantial rates of resistance to four other antibiotics, and these instances also showed a high degree of association with penicillin resistance. There was no correlation between the occurrence of penicillin resistance and the length of time of administration of penicillin in prophylactic doses. It is suggested that the high rate of occurrence of penicillin-resistant staphylococci among these children is due to the continued administration of penicillin, and probably reflects maintenance of penicillin-resistant, phage III strains which the patients may have harbored on discharge from hospital wards.

VIABILITIES OF FROZEN MAMMALIAN CELLS FOLLOWING SLOW THAWING.

Summary 1. Under the conditions described, good viability of frozen mammalian cell cultures was often obtained following slow thawing in the presence of added di-methylsulfoxide. 2. Variation of pH of the freeze medium had no effect on viabilities of frozen HeLa, SJ. cells following slow or rapid thawing. 3. Dimethylsulfoxide is superior to glycerol in preventing damage of the cells tested during freezing. This is particularly evident when slow thawing is employed.